Antiproliferative and Apoptotic Effects of Butyrolactone Lignans from Arctium lappa on Leukemic Cells

• Abstract
• Materials and Methods
• Acknowledgements
• References

Abstract

In the course of screening for pharmacologically active substances from extracts of crude drugs used traditionally in Sino-Japanese herbal medicines, it was found that the 70 % ethanol extract from the fruits of Arctium lappa L. (Compositae) showed potent antiproliferative activity against B cell hybridoma cell, MH60. By bioassay-guided purification, a new lignan, (+)-7,8-didehydroarctigenin, together with the known lignans (-)-arctigenin and (-)-matairesinol were isolated as the active ingredients from an aqueous ethanolic extract of the fruits of A. lappa. Of these active compounds, (-)-arctigenin showed the most potent antiproliferative activity against MH60 cells (IC₅₀ : 1.0 μM), and the activity was suggested to be due to apoptosis.

In the screening of pharmacologically active substances from Sino-Japanese herbal medicines, potent antiproliferative activity against B cell hybridoma cells, MH60, was measured in the extract of Arctium lappa L. (Compositae). Bioassay-guided purification of an aqueous ethanolic extract of the fruits of A. lappa led to the isolation of compounds 1 - 3 (Fig. [1]). By analysis of their spectroscopic data (HR-FAB-MS, EI-MS, ¹H-¹H COSY, HMQC, HMBC, DEPT and NOE-NMR) and comparison of those data to previously reported values [1], [2], [3], [4], [5], [6], compounds 1 and 2 were identified as (-)-arctigenin and (-)-matairesinol. The ¹H- and ¹³C-NMR spectra of compound 3 were similar to those of (-)-arctigenin (1). However, the H-7 methylene and H-8 methine proton signals were absent, and H-7 methine (δ_C = 137.42, δ_H = 7.53) and a quaternary carbon signal (δ_C = 125.66) were observed in 3, suggesting the presence of a double bond. The molecular formula of 3 was determined by HR-EI-MS as C₂₁H₂₂O₆ and coincided with the data of ¹H- and ¹³C-NMR analyses. In addition to the above spectral evidence, all the assignments of 3 were in accordance with the data of the previously reported lignan, (-)-7,8-didehydroarctigenin [7], except for the optical rotation value. Compound 3 showed a positive optical rotation value, [α]_D: + 16.8 (MeOH; c 0.1). Thus, compound 3 was identified as a new lignan, (+)-7,8-didehydroarctigenin, which is an enantiomer of (-)-7,8-didehydroarctigenin [7]. Copies of the original spectra are obtainable from the author of correspondence. Of these active compounds, (-)-arctigenin (1) showed the highest biologocal activity with an IC₅₀ value of 1.0 μM against MH60 cells. The IC₅₀ values of (-)-matairesinol and (+)-7,8-didehydroarctigenin were 8.4 and 13.5 μM, respectively (Fig. [2] a). Since MH60 is an IL-6-dependent cell, an inhibitor of IL-6 activity, madindoline A [8], was used as a positive control, and the IC₅₀ value of madindoline A was 8 μM (Fig. [2] a). Although some variations of the activity between tested cell lines were observed, these isolated compounds showed antiproliferative activity not only against MH60 cells, but also against TF-1 and SP-2 cells (Figs. [2] b and [2] c), suggesting that the antiproliferative effect was not due to an inhibition of IL-6 activity. Because of its yield and activity, the following experiment was performed using (-)-arctigenin.

In order to investigate whether the cell growth inhibition and cytotoxic activities were caused by apoptosis, DNA fragmentation was analyzed with the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) assay. TUNEL-positive cells were observed after treatment with (-)-arctigenin (Figs. [3] a and [3] b). (-)-Arctigenin-induced cell death was significantly inhibited by the pan-caspase inhibitor, Z-VAD-FMK (Fig. [3] c). This suggests caspase-mediated apoptotic cell death.

Materials and Methods

The fruits of A. lappa L. were purchased from Tochimoto Co. Ltd. (Osaka, Japan), and a voucher (No. 210 200) has been deposited at the Herbarium of Oriental Medicine Research Center, the Kitasato Institute. Recombinant murine IL-6 and recombinant human IL-5 were from Genzyme Inc. (Cambridge, MA, USA). The broad-spectrum caspase inhibitor, Z-VAD-FMK, was obtained from MBL (Nagoya, Japan). MH60 cells, which are an IL-6-dependent murine B cell hybridoma, were supplied by Prof. Dr. T. Hirano, Osaka University, and TF-1 cells, which are an IL-5-dependent human myeloid erythroleukemia, and SP-2 cells, which are a murine myeloma, were supplied by Prof. Dr. K. Takatsu, University of Tokyo. These cells were maintained in RPMI 1640 medium supplemented with 10 % FBS, 100 IU/mL penicillin, 100 μg/mL streptomycin and 5 × 10 ^{- 5} M 2-mercaptoethanol (RPMI 1640 - 10 % FBS). For culturing of MH60 and TF-1 cells, 8 U/mL IL-6 and 5 U/mL of IL-5 each were added into the culture media, respectively. Madindoline A obtained from Streptomyces sp. [8] was kindly provided by Prof. Dr. S. Omura (the Kitasato Institute) and was used as a positive control.

The cells were seeded in 96-well microplates at a density of 1 × 10⁴ cells/well and then cultured with or without test samples for 3 days. Then, the cell proliferation was measured with a fluorometric assay, Alamar Blue^TM reduction assay [9]. The fluorescence intensity caused by viable cell was measured with Fluoroskan II (Labsystems Oy, Helsinki, Finland) at an excitation wavelength of 544 nm and an emission wavelength of 590 nm.

The pulverized fruits of A. lappa (100 g) were extracted with 70 % EtOH under reflux (1 L, 2 h, twice). The extract solutions were filtered and then evaporated at 40 °C under reduced pressure. The EtOH extract was resuspended in 500 mL water and then partitioned twice with the same volume of n-hexane. The resulting aqueous layer was partitioned three times with the same volume of AcOEt. The aqueous layer was further successively partitioned with n-BuOH. Each fraction was concentrated to give an n-hexane fr. (1.23 g), AcOEt fr. (6.59 g), n-BuOH fr. (7.26 g), and H₂O fr. (4.37 g), respectively. At a concentration of 5 μg/mL, the antiproliferative activity of n-hexane fr., AcOEt fr., n-BuOH fr., and H₂O fr. were 5.5 %, 65.6 %, 36.4 % and 0 %, respectively. The AcOEt fr. was chromatographed on a silica gel (Wakogel C200, 500 g) and eluted in a stepwise manner with each 1.5 L of CHCl₃-MeOH (1 : 0, 19 : 1, 9 : 1, 4 : 1, 1 : 1, 0 : 1) mixtures to obtain 6 fractions (Fr. I to Fr. VI). Of these fractions, Fr. I exhibited antiproliferative activity, and the activity at a concentration of 4 μg/mL was 98.5 %. Fr. I was further subjected to reversed phase HPLC column chromatography (Cosmosil C₁₈, 10 × 250 mm), 60 - 80 % MeOH linear gradient elution (30 min) at a flow rate of 3.0 mL/min, to afford compounds 1 (t_R = 8.8 min, 70 mg), 2 (t_R = 7.2 min, 10.8 mg) and 3 (t_R = 10.0 min, 4.2 mg).

(+)-7,8-Didehydroarctigenin (3): Colorless resin; [α]_D ²⁰: + 16.8° (MeOH; c 0.1); ¹H-NMR (CDCl₃): δ = 7.53 (1H, d, J = 2.0 Hz, H-7), 7.22 (1H, dd, J = 2.0, 8.0 Hz, H-6), 7.03 (1H, d, J = 2.0 Hz, H-2), 6.99 (1H, d, J = 8.0 Hz, H-5), 6.81 (1H, d, J = 8.0 Hz, H-5′), 6.74 (1H, dd, J = 2.0, 8.0 Hz, H-6′), 6.67 (1H, d, J = 2.0 Hz, H-2′), 5.9 (1H, br s, 4-OH), 4.28 (2H, d, J = 4.0 Hz, H-9′), 3.92 (3H, s, 3-OCH₃), 3.86 (6H, s, 3′ and 4′-OCH₃), 3.83 (1H, m, H-8′), 3.08 (1H, dd, J = 4.2, 14.5 Hz, H-7′), 2.65 (1H, dd, J = 10.0, 14.5 Hz, H-7′); ¹³C-NMR: δ = 172.68 (C-9), 149.15 (C-3′), 148.13 (C-4′), 147.61 (C-3), 146.72 (C-4), 137.42 (C-7), 130.44 (C-1′), 126.55 (C-1), 125.66 (C-8), 123.93 (C-6), 120.70 (C-6’), 114.96 (C-5), 112.69 (C-2), 112.32 (C-2′), 111.55 (C-5′), 69.70 (C-9′), 56.00 (3-OCH₃), 55.94 and 55.92 (3′- and 4′-OCH₃), 39.70 (C-8′), 37.32 (C-7′); EI-MS: m/z (rel. int. %) = 370 [M] ⁺ (64), 219 [C₁₂H₁₁O₄]⁺ (33), 151 [C₉H₁₁O₂]⁺ (100), 131 (31), 107 (32); HR-EI-MS: m/z = 370.1421, calcd. for C₂₁H₂₂O₆ (M⁺): 370.1426.

¹H-NMR and ¹³C-NMR spectra were obtained on a Varian XL-400 spectrometer operating at 400 MHz and 100 MHz, respectively. FAB- and EI-MS were recorded on JMS-DX 300 and JMS-AX505 HA mass spectrometers. Optical rotation was measured on a JASCO DIP-370 digital polarimeter. TUNEL staining was performed with a DeadEnd^TM Fluorometric TUNEL System (Promega, Madison, MI, USA), and analyzed by a COULTER EPICS ELITE flow cytometer.

Acknowledgements

We would like to thank Ms. A. Nakagawa, Ms. C. Sakabe and Ms. M. Sato for spectroscopic analysis, and Ms. S. Tanaka for technical assistance. A part of this work was supported by a grant of the 21st Century COE Program from the Ministry of Education, Culture, Sports, Sciences, and Technology of Japan.

References

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• 7 Marco J A, Sanz-Cervera J F, Garcia-Lliso V, Susanna A, Garcia-Jacas N. Sesquiterpene lactones, lignans and aromatic esters from Cheirolophus species.  Phytochemistry. 1994;  37 1101-7
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• 8 Hayashi M, Kim Y P, Takamatsu S, Enomoto A, Shinose M, Takahashi Y. et al . Madindoline, a novel inhibitor of IL-6 activity from Streptomyces sp. K93 - 0711. I. Taxonomy, fermentation, isolation and biological activities.  J Antibiot. 1996;  49 091-5
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Prof. Dr. H. Yamada

Kitasato Institute for Life Sciences

Kitasato University

5-9-1 Shirokane

Minato-ku

Tokyo 108-8641

Japan

Fax: +81-3-5791-6121

Email: yamada@lisci.kitasato-u.ac.jp

References

• 1 Eich E, Perts H, Kaloga M, Schulz J, Fesen M R, Mazumder A. et al . (-)-Arctigenin as a lead structure for inhibitors of human immunodeficiency virus type-1 integrase.  J Med Chem. 1996;  39 86-95
  CrossrefPubMedSearch in Google Scholar
• 2 Youssef D, Frahm A W. Constituents of the Egyptian Centaurea scoparia; III. Phenolic constituents of the aerial parts.  Planta Med. 1995;  61 570-3
  PubMedSearch in Google Scholar
• 3 Rhaman M MA, Dewick P M, Jackson D E, Lucas J A. Lignans of Forsythia intermedia .  Phytochemistry. 1990;  29 1971-80
  CrossrefPubMedSearch in Google Scholar
• 4 Christensen L P, Lam J. Acetylenes and other constituents from Centaurea species.  Phytochemistry. 1991;  30 3289-92
  CrossrefPubMedSearch in Google Scholar
• 5 Inagaki I, Hisada S, Nishibe S. Lignans of Trachelospermum asiaticum var. intermedium. I. Isolation and structures of arctiin, matairesinoside and tracheloside.  Chem Pharm Bull. 1972;  20 2710-8
  PubMedSearch in Google Scholar
• 6 Ozawa S, Davin L B, Lewis N G. Formation of (-)-arctigenin in Forsythia intermedia .  Phytochemistry. 1993;  32 643-52
  CrossrefPubMedSearch in Google Scholar
• 7 Marco J A, Sanz-Cervera J F, Garcia-Lliso V, Susanna A, Garcia-Jacas N. Sesquiterpene lactones, lignans and aromatic esters from Cheirolophus species.  Phytochemistry. 1994;  37 1101-7
  CrossrefPubMedSearch in Google Scholar
• 8 Hayashi M, Kim Y P, Takamatsu S, Enomoto A, Shinose M, Takahashi Y. et al . Madindoline, a novel inhibitor of IL-6 activity from Streptomyces sp. K93 - 0711. I. Taxonomy, fermentation, isolation and biological activities.  J Antibiot. 1996;  49 091-5
  PubMedSearch in Google Scholar
• 9 Pag B, Pag M, Noël C. A new fluorometric assay for cytotoxicity measurements in vitro .  Int J Oncol. 1993;  3 473
  PubMedSearch in Google Scholar

Prof. Dr. H. Yamada

Kitasato Institute for Life Sciences

Kitasato University

5-9-1 Shirokane

Minato-ku

Tokyo 108-8641

Japan

Fax: +81-3-5791-6121

Email: yamada@lisci.kitasato-u.ac.jp